Transsynaptic Coordination of the Formation of Morphofunctional Contacts between the Brain and the Neurotransplant: an Ulrastructural Study.

We studied the role of neurotransmitter signaling mediated by synaptic vesicles in the formation of aberrant functional connections between fascia dentata grafts and the somatosensory neocortex in adult rats. Quantitative analysis of the different populations of synaptic vesicles in the ectopic giant axonal endings of granular neurons was performed and the results were compared with the normal. Two pools of small clear vesicles (rapidly releasable pool and pool of reserve vesicles circulating in the active zone) and one pool of large dense-core vesicles were analyzed. Significant differences from the control suggest that synaptic integration of the transplants into the recipient brain is coordinated by transsynaptic signaling and mediated by different populations of synaptic vesicles.

Structural signs of dynamic state of synaptic contacts between neurotransplant and brain.

We studied ultrastructure of synaptic connections between long-living dentate fascia transplants and somatosensory area of the neocortex in rats. Giant synaptic terminals of granular neurons upon contact with unusual neuronal targets in the neocortex reproduced their specific constitutive morphological features. At the same time, developing synapses with signs of active structural and metabolic reorganization were revealed. This is indicative of a dynamic state and instability of functional connections between the transplant and brain despite long time after transplantation.

[Participation of puncta adherentia junctions in the formation of synaptic connections between a dentate fascia graft and the host brain].

The fetal dentate fascia of Wistar rats on the 20th day of gestation was heterotopically grafted into the somatosensory neocortex of adult rats. Granule cells of a graft projected their axons (mossy fibers) to the host brain and established synaptic contacts with inappropriate targets. The organization of ectopic mossy fiber synapses was studied by electron microscopy. It was shown that ectopic synapses reproduce the structural determinants of hippocampal giant synapses and induce a subcellular reorganization of postsynaptic neocortex dendrites. Using morphometric analysis, a significant increase was found in the number of discrete puncta adherentia junctions and their total length in ectopic synapses as compared with the control group. The data obtained indicate that puncta adherentia contacts participate in the structural and chemical adaptation of neuronal targets to alien axons growing from transplants.

Involvement of neuropeptide mechanisms in the process of integration of heterotopic dental fascia transplants with recipient brains.

Embryonic dentate fascia was transplanted into the somatosensory area of the neocortex of adult rats. Ultrastructural and morphometric analyses of giant synapses formed by the granule neurons of transplants with inappropriate neuronal targets in the recipient brains were performed after nine months. As compared with intact synaptic terminals in the control hippocampus, there were differences in the quantity and distribution of large synaptic vesicles with electron-dense centers storing neuropeptide cotransmitters. The proportion of peptidergic vesicles (of the total number of vesicles) in ectopic giant synapses was 5.8 +/- 0.6%, compared with 3.3 +/- 0.6% in controls. Accumulations of large, dense vesicles close to the active zones of aberrant connections were seen almost 7.9 times more often than in controls. These results show that neuropeptide transmitters are critical for maintaining synaptic connections between heterotopic dentate fascia transplants and recipient brains.

[Involvement of neuropeptide mechanisms in the integration of heterotopic dentate fascia grafts and recipient brain].

Embryonic dentate fascia was grafted into the somatosensory neocortex of adult rats. Nine months post-grafting, the ultrastructural and morphometric analysis of the giant synapses established between the grafted granular neurons and inappropriate targets in the recipient brain was performed. As compared to the intact synaptic endings in the control hippocampus, differences were found in both the number and distribution of large dense-core synaptic vesicles, which store the neuropeptide co-transmitters. The peptidergic vesicle proportion (of total vesicle pool) within the ectopic giant synapses was 5.8 +/- 0.6% (versus 3.3 +/- 0.6% in the control). Clusters of large dense-core vesicles near the active zones of aberrant connections were observed almost 7.9 times more frequently than that of normal contacts. These data provide evidence that neuropeptide transmitters are critical for the maintenance of synaptic connections between the heterotopic dentate grafts and host brain.

Genetically engineered cells with regulatable GABA production can affect afterdischarges and behavioral seizures after transplantation into the dentate gyrus.

Intractable seizures originating in the mesial temporal lobe can often be controlled by resection. An alternative to removing hippocampal tissue may be transplantation of GABA-producing cells. Neural cell transplantation has been performed in hundreds of patients, including some with temporal lobe epilepsy. This study evaluates the seizure-suppressing capabilities of engineered GABA-producing cells transplanted into the dentate gyrus. Immortalized neurons were engineered to produce GABA under the control of doxycycline. The cells were characterized for GABA production in vitro and for their ability to raise GABA concentrations in vivo. Cells were transplanted bilaterally into the dentate gyrus of rats and tested in two separate paradigms. Afterdischarge thresholds and durations were tested with granule cell stimulation, and the development of behavioral seizures, induced by daily electrical stimulation of the major excitatory input pathway into the dentate gyrus, was assessed in the presence, or the absence, of doxycycline. GABA production was under the tight control of doxycycline. Cells engineered to produce GABA raised tissue GABA concentrations in the hippocampus compared with non GABA-producing cells, and this was abolished when doxycycline was administered. GABA-producing cells raised the threshold, and shortened the duration of hippocampal afterdischarges elicited by granule cell stimulation. Lastly, the appearance of stage 5 seizures was slowed in the kindling paradigm, compared with a group that received non-GABA-producing cells, and compared with a group that received GABA-producing cells but was administered doxycycline. This study shows that targeted hippocampal implants of genetically engineered cells have the potential to raise GABA levels and to affect seizure development. The ability to suppress the production of GABA, and to modulate the physiological effects of the transplanted cells provides an important level of experimental control. These techniques, combined with stem cell technology, may advance cell-based therapies for epilepsy and other diseases of the CNS.

Heterotypic neuronal differentiation of adult subependymal zone neuronal progenitor cells transplanted to the adult hippocampus.

Neuronal progenitor cells (NPCs) residing in the adult subependymal zone (SEZ) are a potential source of expandable cells for autologous transplantation to replace neurons lost in multiple types of brain injury. To characterize the capacity of these cells for neuronal differentiation in a mature ectopic environment, NPCs expanded from the SEZ of adult rats were transplanted to the adult dentate gyrus. Cultures comprised a heterogeneous population of proliferating cells, which expressed nestin (47%) and GFAP (37%), with many cells expressing both progenitor cell markers (31%). In grafts of undifferentiated cells, as well as in grafts of cells that were induced to differentiate in vitro with retinoic acid, 35% of the transplanted SEZ-derived cells exhibited immunohistochemical and morphological features characteristic of hippocampal granule cell neurons. These novel results indicate that in vitro expanded adult SEZ NPCs are capable of heterotypic neuronal differentiation in a neurogenic region of the adult brain.

[Synaptic contacts of neurons of fascia dentata transplants with nonspecific targets in neocortex of recipients]. / Sinapticheskie kontakty neironov transplantatov zubchatoi fastsii s nespetsificheskimi misheniami v neokortekse retsipientov.

We carried out an electron microscopy study of possible synaptic contacts of the neurons of intracortical transplants of the rat brain fascia dentata with targets in the recipient somatosensory cortex. The axons of fascia dentata granular cell and their synaptic terminals could be easily identified in the neocortex due to their distinct morphological features (mossy fibers), although the fascia dentate cells normally do not interact with the neocortex. Thin nonmyelenized mossy fibers were found in both an intermediate zone between the transplant and brain and in the adjacent brain. Their presynaptic buds, like in situ, had large size and formed characteristic terminal, intraterminal, and en passant multiple synaptic contacts and desmosome-like junctions. The aberrant nerve fibers used perykaryons, dendrites of varying diameter, and dendrite spikes of the somatosensory cortex pyramidal neurons as postsynaptic targets in the neocortex. In addition to vacant spaces that appeared in the brain as a result of transplantation, the ingrowing axons induced the formation of additional contact sites: deep invaginations of the plasmalemma of perykaryons, somatic spikes, terminal branchings of dendrites, and dendritic outgrowths of complex branched shape. These aberrant contacts were characterized by the presence of polyribosomes, endoplasmic reticulum cisternae, and mitochondria in the postsynaptic loci. Osmiophility and extension of desmosome-like junctions were also enhanced in such synapses. Thus, it was shown that mossy fibers ingrowing in the recipient neocortex were capable of forming cell-to-cell contacts with signs of functional synapses to atypical cell targets.

[Developmental neuronal plasticity and its importance in the recovery and function of neural tissue]. / Vývojová neuroplasticita a její význam pro obnovu struktury a funkce nervové tkáne.

Plasticity is understand as a natural ability of the nervous tissue to respond to intrinsic and extrinsic stimuli by means of functional or structural changes. All its manifestations, including the developmental changes and processes of recovery are controlled by general genetic programs. One of the major research goals in the field of neuroplasticity is to design a strategy for the activation of the functional recovery. Enough evidence has shown that the internal environment of the nervous tissue is one of the decisive factors which determine the degree of both the developmental and regenerative plasticity. To improve capacity for the recovery it is therefore necessary to activate these intrinsic neuroplastic mechanisms. One alternative is the method of implantation of the embryonal nerve cells suspension into the site of neuronal lesion. The grafted cells can either replace the lost cells or they might become a source of stimulating factors which may activate the process of recovery. In the model of the intrahippocampal pathways interruption (experimental partial elimination of granule cells in the dorsal blade of the dentate gyrus), the subsequent implantation of embryonal neurons of the same origin into the site of lesion can partly restore the structural pattern of the gyrus dentatus and probably preserve some of the afferent connections. However, the outgrow of efferent fibers probably remains limited to the grafted tissue.

[Differentiation of neurons and synapses of mossy fibers in transplants of fascia dentata developing in the rat neocortex]. / Differentsirovka neironov i sinapsov mshistykh volokon v transplantatakh zubchatoi fastsii, razvivaiushchikhsia v neokortekse krys.

Embryonic fascia dentata tissue isolated from the hippocampus was transplanted heterotopically into the neocortex of adult rats. Ultrastructural characteristics of neurons and synapses in transplants were studied nine months later. It has been found that the main types of neurons present in fascia dentata undergo differentiation in the transplants, and a dense neuropile containing various types of synapses is produced. A characteristic feature of the transplanted neurons is the presence of additional microspines on somatic and dendrite surfaces; this appears to be due to a deficiency of external and internal afferents. Gigantic synaptic terminals of granule cell axons (mossy fibers) in transplants possess unique morphological characteristics, which allow their identification in a complex neuropile. Just as in situ, they form two types of contacts: chemical asymmetric contacts with dendrite spines and desmosome-like ones with dendrite surface characteristics. However, accumulations of large vesicles with electron-dense centers can often be observed near the active zones of the synapses, and desmosome-like connections are more prominent. The most important feature is that gigantic synapses in transplants use midsize and small dendrites as postsynaptic targets up to terminal branches, and they contact with spines of the usual shape and size, whereas in situ terminal synaptic contacts of mossy fibers are formed only with gigantic processes of initial segments of the large apical dendrites. Thus, in the absence of normal synaptic targets, mossy fibers can produce contacts having all features of functional synapses, but with atypical postsynaptic structures.